An antibody binds to an antigen and neutralizes it by preventing it from binding to its endogenous target (e.g. receptor or ligand) or by inducing effector responses that lead to antigen removal. To efficiently remove and/or destroy antigens foreign to the body, an antibody should exhibit both high affinity for its antigen and efficient effector functions. Antibodies having multispecificities (such as, for example, bispecific antibodies) are useful for mediating complementary or synergistic responses of multiple antigens.
Antibody effector functions are mediated by an antibody Fc region. Effector functions are divided into two categories: (1) effector functions that operate after the binding of antibody to an antigen (these functions involve the participation of the complement cascade or Fc receptor (FcR)-bearing cells); and (2) effector functions that operate independently of antigen binding (these functions confer persistence of antibody in the circulation and its ability to be transferred across cellular barriers by transcytosis). See, for example, Ward and Ghetie, 1995, Therapeutic Immunology 2:77-94. Interactions of antibodies and antibody-antigen complexes with cells of the immune system cause such responses as, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) (reviewed in Daëron, 1997, Annu. Rev. Immunol. 15:203-234; Ward et al., supra; Ravetch et al., 1991, Annu. Rev. Immunol. 9:457-492; and Ravetch et al, 2000, Science 290:84-89.
Because Fc receptors mediate antibody effector function by binding to the Fc region of the receptor's cognate antibody, FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors specific for IgG antibodies are referred to as FcγR; Fc receptors for IgE antibodies are FcεR; Fc receptors for IgA antibodies are FcαR, and so on.
Three subclasses of FcγR have been identified: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). Each FcγR subclass is encoded by two or three genes that undergo alternative RNA spicing, thereby leading to multiple transcripts and the existence of a broad diversity in FcγR isoforms. The three genes encoding the human FcγRI subclass (FcγRIa, FcγRIb, and FcγRIc) are clustered in region 1q21.1 of the long arm of chromosome 1; the genes encoding human FcγRII isoforms (FcγRIIa, FcγRIIb and FcγRIIc) are in region 1q23−24; and the two genes encoding human FcγRIII (FcγRIIIa and FcγRIIIb) are clustered in region 1q22. FcγRIIC is formed from an unequal genetic cross over between FcγRIIA and FcγRIIB, and consists of the extracellular region of FcRIIB and the cytoplasmic region of FcγRIIA.
FcγRIIA encodes a transmembrane receptor FcγRIIA1. Alternative RNA splicing results in FcγRIIA2 that lacks the transmembrane region. Allelic variants of the FcγRIIA gene give rise to high responder (HR) or low responder (LR) molecules that differ in their ability to bind IgG. The HR and LR FcγRIIA molecules differ in two amino acids corresponding to positions 27 and 131. FcγRIIB encodes splice variants FcγRIIB1, FcγRIIB2 and FcγRIIB3. FcγRIIB1 and FcγRIIB2 differ by a 19 amino acid insertion in the cytoplasmic domain of FcγRIIB1; FcγRIIB3 is identical to FcγRIIB2, but lacks information for the putative signal peptidase cleavage site.
The receptors are also distinguished by their affinity for IgG. FcγRI exhibit a high affinity for IgG, Ka=108−109M−1 (Ravetch et al., 2001, Ann. Rev. Immunol. 19:275-290) and can bind monomeric IgG. In contrast, FcγRII and FcγRIII show a relatively weaker affinity for monomeric IgG Ka≦107M−1 (Ravetch et al., supra), and only interact effectively with multimeric immune complexes. The different FcγR subtypes are expressed on different cell types (reviewed in Ravetch, J. V. et al, Annu. Rev. Immunol. 9:457-492). For example, only FcγRIIIA is expressed on NK cells. Binding of antibodies to this receptor leads to ADCC activity typical of NK cells. Human FcγRIIIB is found only on neutrophils, whereas FcγRIIIA is found on macrophages, monocytes, natural killer (NK) cells, and a subpopulation of T-cells. On the other hand, FcγRII receptors with low affinity for monomeric IgG are the most widely distributed FcRs, and are usually co-expressed on the same cells. FcγRII (encoded by CD32) is expressed strongly on B cells, monocytes, granulocytes, mast cells, and platelets, while some T cells express the receptor at lower levels (Mantzioris, B. X. et al., 1993, J. Immunol. 150:5175-5184; and Zola, H. et al., 2000, J. Biol. Regul. Homeost. Agents, 14:311-316). For example, human FcγRIIB receptor is distributed predominantly on B cells, myeloid cells, and mast cells (Ravetch J. V. and et al., 2000, Science 290:84-89).
FcγRIIA and FcγRIIB isoforms contain very similar extracellular domains (approximately 92% amino acid sequence identity) but differ in their cytoplasmic regions, leading to functional differences as “activating receptors” (FcγRIIA) and “inhibitory receptors” (FcγRIIB). FcγRI and FcγRIII receptors also function as activating receptors. These activating receptors contain a 19 amino acid immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic domain. The ITAM sequences trigger activation of src and syk families of tyrosine kinases, which in turn activate a variety of cellular mediators, such as P13K, PLCγ, and Tec kinases. The net result of these activation steps is to increase intracellular calcium release from the endoplasmic reticulum stores and open the capacitance-coupled calcium channel, thereby generating a sustained calcium response. These calcium fluxes are important for the exocytosis of granular contents, stimulation of phagocytosis, ADCC responses, and activation of specific nuclear transcription factors.
Cellular responses mediated by activating FcγRs are regulated by the inhibitory FcγRIIB receptor in the maintenance of peripheral tolerance, regulation of activation response thresholds, and ultimately in terminating IgG mediated effector stimulation (Ravetch, J. V. et al, Annu. Rev. Immunol. 19:275-290 (2001)). Such regulation is initiated by crosslinking of activating receptors with inhibiting FcγRIIB receptors via an antigen-IgG antibody immune complex (See, for example, Ravetch, J. V. et al., 2000, supra). Crosslinking of an ITAM-containing activating receptor leads to tyrosine phosphorylation within the 13 amino acid immunoreceptor tyrosine-based inhibition motif (ITIM) in the FcγRIIB cytoplasmic domain. This “activation” of FcγRIIB initiates recruitment of a specific SH2-containing inositol polyphosphate-5-phosphatase (SHIP). SHIP catalyzes the hydrolysis of the membrane inositol lipid PIP3, thereby preventing activation of PLCγ and Tec kinases and abrogating the sustained calcium flux mediated by influx of calcium through the capacitance-coupled channel. While FcγRIIB negatively regulates ITAM-containing activating receptors (Daëron, M. et al., 1995, Immunity 3:635-646), it has also been shown to negatively regulate receptor-tyrosine kinase (RTK) c-kit in the control of RTK-mediated-mediated cell proliferation (Malbec, O. et al., 1999 J. Immunol. 162:4424-4429).
Antibodies that bind FcγRII receptors have been described in: Looney et al., (1986) J. Immunol. 136:1641-1647; Zipf et al., (1983) J. Immunol. 131:3064-3072; Pulford et al., (1986) Immunology 57:71-76; Greenman et al., (1991) Mol. Immunol. 28:1243-1254; Ierino et al., (1993) J. Immunol. 150:1794-1803. Weinrich et al., (1996) Hybridoma, 15:109-116; Sonderman et al., (1999) Biochemistry, 38:8469-8477; Lyden, T. W. et al. (2001) J. Immunol. 166:3882-3889; and International Publication No. WO 2004/016750, published Feb. 26, 2004. The high-affinity IgER1 receptor, FcεRI, mediates signaling for antigen induced histamine release upon binding of IgE during, for example, allergic reaction (von Bubnoff, D. et al., (2003) Clinical & Experimental Dermatology. 28(2):184-187). FcγRIIB receptors have been shown to interact with and inhibit the activity of FcεRI through the FcγRIIB ITIM domain (Daeron, M. et al. (1995) J. Clin. Invest. 95:577-585; Malbec, O. et al. (1998) J. Immunol. 160:1647-1658); and Tam, S. W. et al. (2004) Allergy 59:772-780). Antibodies that specifically bind human FcγRIIB are needed, not only for research, but also to manipulate FcγRIIB and FcεRI activity to treat disease.